Project Summary The goal of this application is to develop a novel laser-enhanced NMR technology for the highly sensitive atomic-resolution analysis of amino acids, polypeptides and proteins in solution, down to sub-micromolar concentration. The instrumentation involved in this work comprises high-power lasers (both continuous-wave and pulsed) coupled, via fiber optic, to a commercial 600 MHz NMR spectrometer equipped with a quadruple- resonance (HFCN) cryogenic probe. Transient electronic absorption measurements will also be performed to monitor the photoexcited triplet lifetime of photo-CIDNP dyes. The advances gained via this research will enable the high-resolution NMR analysis of polypeptides and proteins at unprecedentedly low concentration. The proposal further extends key method-development originally carried out in the PI?s laboratory on single amino acids to the realm of polypeptides and proteins. We will accomplish the above goals within three steps. First (Specific Aim #1), photo-CIDNP will be explored in the presence of a newly acquired cryogenic probe at 600 MHz, to further extend NMR sensitivity in solution to the nanomolar range. Our studies will employ the recently developed photosensitizer fluorescein, tailored to low-concentration photo-CIDNP, and will take advantage of thorough oxygen depletion in NMR samples, and will employ ultrafast femtosecond laser irradiation for laser-driven NMR sensitivity enhancement. Second (Specific Aim #2), we will systematically extend photo-CIDNP in solution, by extending its applicability to polypeptides, and proteins in simple buffered solution and in more complex highly crowded physiologically relevant environments. We will accomplish the above goal by testing new photosensitizer dyes tailored to optimal performance in the presence of proteins, by developing appropriate multidimensional photo-CIDNP NMR pulse sequences, and by collecting data in buffered solution, cell-free systems, and possibly live bacterial cells. Third (Specific Aim #3), we will extend photo-CIDNP to non-aromatic amino acids by implement NOE modules within photo-CIDNP pulse sequences to spread the photo-CIDNP-enhanced magnetization of polypeptides and proteins from aromatic residues to other amino acids. Finally, we will pioneer new double-laser-irradiation experiments for the transient oxidation of the carbonyl functional group of proteins, which is present in all amino acids, and is known to be particularly oxidizing in its photo-excited state. Once the proposed new technology has been developed, it will immediately become possible to collect hyperpolarized NMR data in solution on amino acids, polypeptides and proteins of biomedical relevance at sub-micromolar concentration in solution and cell-like environments.